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Batman and his sidekick Robin take on The Riddler as he attacks the Gotham Gold Reserves, The Penguin as he plans to control Gotham using penguin robots and The Joker as he intends to destroy the Cathedral and spread his laughing gas throughout Gotham.

Once some of Batman's most fearsome enemies escaped Arkham Asylum and teamed up, Batman and Robin headed towards a bank which had been robbed by The Riddler and Clayface. The Dynamic Duo made their way through the streets defeating the Riddler Goons and Henchmen who tried to stop them. At the bank, a fight ensued against Clayface, who was eventually knocked back into a cage in the vault when the security system pumped poisonous fumes into the air. Two-Face arrived in his Armoured Truck to rescue The Riddler and a giant key he'd stolen. Batman was unable to stop his escape, but did manage to plant a tracking device on the truck.

Using the tracking device, Batman and Robin followed the truck to Mr. Freeze's old Ice Cream Factory. The duo was immediately attacked by more Riddler Goons and Henchmen, but once they entered the factory, they encountered a new enemy, the Freeze Girls. By reaching the end of the factory, the duo encountered Mr. Freeze and managed to defeat him. The Riddler escaped once again in Two-Face's armoured truck, this time with a Freeze Ray.

Batman gave chase to Two-Face's armoured car in the Batmobile, successfully destroying it. Two-Face and Riddler went in different directions to try and escape. Riddler was cornered against Police Headquarters. Riddler tried to fend of by making desperate attempts at the duo with his "?" cane, but he was rescued by Poison Ivy.

In the Batcave, Batman used the Batcomputer to analyze a leaf from the vine Ivy used to save the Riddler. He discovered it was from the Botanical Gardens. Batman and Robin reached the gardens to discover the Janitors and Scientists under siege from not only The Riddler's goons and henchmen, but from humanoid plant creatures, presumably created by Poison Ivy. In a closed off area behind the garden's laboratory, Batman and Robin discovered and defeated Poison Ivy, but The Riddler escaped yet again (this time with mutant vine seeds) when he pointed out to Batman that Ivy caused Robin to fall in love with her. Batman forced Robin back to the batcave, while Ivy was arrested.

Back in the Batcave, with help from Alfred, Batman and Robin solved the clues The Riddler had left at each of his escapes and discovered his plot was to stage a siege on the Gotham Gold Reserves. And sure enough, inside the Gold Reserves, they found Riddler and Two-Face. Two-Face created a diversion by shooting a nearby barrel of toxic waste onto the floor while The Riddler continued on. Using the Attract Suit, Robin created a swamp boat in which he and Batman used to reach Two-Face. A fight ensured in which Two-Face was nearly defeated, but he got away at the last moment, calling more henchmen and goons to cover him in his flight.

Batman and Robin followed Two-Face through the vault. Riddler ordered Two-Face to attack. This time, Two-Face was almost defeated, but ran to the side of The Riddler at the last moment. The Riddler then used the vault's defence laser to try and defeat the Dynamic Duo. Batman and Robin used the chromed gold in the vault to deflect the laser beams back at The Riddler until his laser was destroyed. The Riddler then changed his tactics to using the mind control technology in his "?" cane to take control of a whimpering Two-Face, and attack the Duo himself. Eventually, both Riddler and Two-Face were knocked back into a pile of gold bars, subduing them both.

When the Gotham Diamond was stolen from a museum, Batman and Robin found Catwoman fleeing from the scene and gave chase to her on the rooftops of Gotham. They caught up with her, once and a short fight ensued, after which Catwoman escaped. The duo continued to follow her above the city until she was finally cornered. She drew Batman in for a kiss, during which she threw the diamond to the streets bellow. Catwoman was then arrested and placed in a police van.

While in the Batboat, Batman was ambushed by Penguin Goons in submarines. Batman worked his way through the harbour, to find The Penguin's submarine. The submarine was destroyed, Penguin and Killer Croc, (who had helped Penguin escape the museum earlier) headed for the sewers. Batman and Robin caught up with the criminals, but Killer Croc threw the door which gave access to the sewer in Robin's direction to cause a diversion.

Batman and Robin followed the criminals into the sewer where they were attacked by Penguin's Henchmen and Goons. The Duo eventually reached the Police Headquarters, by way of the sewage system, to find Catwoman had been freed and that she had fled the scene with Penguin. Killer Croc was waiting for Batman, and began throwing nearby debris in his direction. When Croc would change his tactics and charge at Batman or Robin, the duo used this time to strike and defeat him. When Croc fell, he dropped a flyer for the Gotham City Zoo.

When Batman and Robin reached the zoo, they discovered the Man-Bat in the hull of a boat Penguin had been using as a lookout. Man-Bat was wrapped in his own wings and defeated.

Batman and Robin continued on to the "Arctic World" portion of the zoo to discover Police Officers had been defeated by a large group of Robotic Penguins. Batman and Robin made their way past the Penguin Minions and the returned Freeze Girls to a cave in the exhibit. In the cave, Batman discovered a satellite dish (which had been stolen by Bane previously) powered by the Gotham Diamond which controlled the Penguin Minions which were assaulting the city. Batman redirected Penguin's bombers into treadmills which gave power to the satellite. Penguin, Catwoman and the Freeze Girls attacked the duo, only to be defeated and returned to Arkham Asylum.

While shopping for a new cape on the Batcomputer, Batman received a transmission of The Joker laughing at his camera. Alfred helped to prepare the Batwing and Bat-copter for the journey to the factory which transformed the Red Hood into the Joker to investigate. In the factory, Batman and Robin were attacked by Joker Goons and Henchmen. In the back of the factory, Batman discovered The Mad Hatter extracting the factory's chemicals. He made attempts to subdue Batman and Robin with his mind-control technology, but failed as he could only control one person as a time.

During this adventure, Joker and Harley Quinn captured Commissioner Gordon and threatened to kill him if Batman did not act. Batman reached the carnival where Gordon was being held, and a fight ensued with Harley Quinn, while The Joker escaped on his helicopter. Gordon was freed and Harley was contained in a Police Van for the time being.
Batman meant to give chase to Joker in his helicopter, but ended up pursuing Scarecrow in his bi-plane. Scarecrow's bi-plane was shot down and crashed into Joker's helicopter in its fall. Both aircrafts were destroyed, and Batman left both villains for dead, not to notice that they survived by clinging onto weather balloons and fell through to the Gotham Art Gallery, where Scarecrow was captured by the Police.

Joker, however, escaped the gallery and met with Killer Moth, who was to help him reach his destination. While on their way, they encountered Gordon, freed Harley Quinn, and were causing havoc on the nearby Police Forces. Batman received a transmission of the trouble on the Batcomputer. He and Robin reached the scene of the crime to find Killer Moth still there. They gave chase to him through the city streets. They eventually cornered and threatened him to tell Batman where Harley and The Joker were, or be sprayed with bug poison. Killer Moth pointed through a hole in the wall he was pinned up against, and Joker's truck was seen pulling up the Gotham City Cathedral.

Batman, Robin, and the remaining Police Forces rushed to the Cathedral. To gain entry, Robin opened up a window of which Harley Quinn jumped out. Batman used the water turrets mounted on the back of police vans to defeat her. Batman and Robin chased Harley to the top of the Cathedral where she and The Joker hid in giant bells. Once they were discovered by Batman, The Joker summoned his helicopter for protection, but once the helicopter was destroyed, it collapsed the platform which The Joker and Harley were on, knocking Harley unconscious. The Joker then attacked the Dynamic Duo, successfully defeating them with his Joy-Buzzer, knocking them unconscious. Harley was revived by the time Batman was. Batman threw a Batarang at the giant bells which disturbed the bats in the tower who attacked Harley and The Joker who were then arrested by Commissioner Gordon and sent back to Arkham Asylum.